1. Field of the Invention
The present invention relates generally to splicing factors and specifically to regulation of splicing and alternative splicing of nuclear messenger RNA precursors by a novel protein serine kinase.
2. Description of Related Art
Most nuclear messenger RNA precursors (pre-mRNA) in higher eukaryotes contain multiple introns which are precisely excised by RNA splicing. Several pre-mRNAs are alternatively spliced in different cell types or at different times during development. Alternative splicing can result in the production of more than one different protein from a single pre-mRNA. One mode of splicing can generate a mRNA that lacks an open translational reading frame and alternative splicing of the same pre-mRNA can yield a functional protein. Alternative splicing has been described in the regulatory hierarchy of sex determination of Drosophila and in many examples of tissue-specific gene expression (Smith, et al., Genet. Eng., 12:139, 1990; Baker, B., Nature, 340:521, 1989). The understanding of the mechanisms of RNA splicing is of fundamental importance in developmental biology.
Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine rich domain (SR proteins) are concentrated in "speckles" in the nucleus of interphase cells (Fu, X.-D. & Maniatis, T. Nature, 343:437-441, 1990). It is believed that nuclear speckles are storage sites, while splicing occurs on nascent transcripts (Spector, D. L. Annu. Rev. Cell Biol. 9:265-315, 1993). Splicing factors redistribute in response to transcription inhibition (Carmo-Fonseca, M., et al., J. Cell Biol. 117:1-14, 1992; O'Keefe, R. T., et al., J. Cell Biol., 124:249-260, 1994), heat shock or viral infection (Jimenez-Garca, L. F., et al., Cell, 73:47-59, 1993), and nuclear speckles break down and reform as cells progress through mitosis (Spector, D. L., et al., EMBO J. 10:3467-3481, 1991; Peter, M. et al., Cell, 61: 591-602, 1990).
The SR family of splicing factors are phosphoproteins that share a phosphoepitope. Phosphorylation and dephosphorylation appear to regulate the activity and intracellular location of these splicing factors. SR proteins are characterized by the presence of RNA binding motifs and the SR domain, which consists largely of arginine/serine repeats. SR proteins contain consensus sequences (S/T*-P-X-R/K; *indicates the phosphorylation site) (SEQ ID NO:8) for the major mitotic kinase p34.sup.cdc2 (Moreno, S., et al., Cell, 61:549-551, 1990). It was believed that p34.sup.cdc2 may directly phosphorylate SR proteins and regulate their localization during the cell cycle. However, the present invention which identifies a novel kinase, SRPK1, renders this incorrect.
The SR proteins play a critical role in the initiation of spliceosome assembly on pre-mRNA substrates. SR proteins can also alter alternative splice site selection by promoting the use of proximal 5' and 3' splice sites in a concentration dependent manner. A number of recent studies suggest that alternative splice site usage is mediated, at least in part, by direct binding of an SR protein to the site, or by recruiting an SR protein to the site by specific alternative splicing regulators. Thus, it appears that alternative splicing may be regulated by controlling the expression and/or activity of SR proteins.
The levels of individual SR proteins have been reported to differ among tissues (Zahler, et al., Science 260:219-222, 1993). However, the quantitation of protein expression depends on a monoclonal antibody that recognizes a specific phosphoepitope present in all SR family members. Thus, the variation in SR proteins detected could reflect the expression levels of SR proteins and/or their differential phosphorylation in different tissues. The SR domain has recently been shown to be responsible for protein-protein interactions during spliceosome assembly (Kohtz, et al., Nature, 368:119-124, 1994; Amrein, et al., Cell 76:735, 1994; Wu, et al., Cell 75:1061-1070, 1993). Since ample potential phosphorylation sites are present in the SR domain, phosphorylation may modulate SR protein interactions.
Two lines of evidence suggest that phosphorylation plays a role in pre-mRNA splicing. First, a U1-snRNP associated kinase activity has been described, that could phosphorylate the SR domain in the SR family member SF2/ASF as well as a similar motif consisting of serine/arginine repeats in the U1-70 kD protein (Woppmann, et al., Nucl. Acids Res. 21:2815-2822, 1993). Incorporation of a nonhydrolysable homolog of ATP into the isolated U1 snRNP particle by the kinase impaired its ability to complement splicing in a U1 snRNP depleted nuclear extract (Tazi, et al., Nature 363:283-286, 1993). Secondly, it has been reported that inhibitors of phosphatases can specifically inhibit splicing in vitro, suggesting that dephosphorylation is required for splicing (Mermoud, et al., Nucl. Acids Res., 20:5263, 1992). Together, these studies show that dephosphorylation is important for splicing. Phosphorylation of SR proteins and other splicing factors containing a similar SR domain may be essential for splicing and the possibility that phosphorylation and dephosphorylation of these proteins occurs during different stages of splicing may also be significant to regulation of splicing of pre-mRNAs.
It is important to find factors which regulate SR proteins, in view of the key role that these proteins play in regulating RNA splicing, since regulation of splicing is fundamental to the growth, differentiation and development of the cell. The present invention provides such a factor in the form of a novel protein which is a cell cycle regulated serine kinase (SRPK1) specific for SR splicing factors. Molecular cloning revealed that SRPK1 is highly related to a C. elegans kinase and the fission yeast kinase dsk1 (Takeuchi, et al., Mole. Biol. Cell, 4:427, 1993). Experimental studies have shown that SRPK1 specifically induced disassembly of nuclear speckles, but had little effect on coiled bodies or other nuclear structures in interphase nuclei. Also, a high level of SRPK1 was inhibitory to splicing in vitro. These observations suggest that SRPK1 is an important regulator of splicing by controlling the intranuclear distribution of splicing factors in interphase cells, and for reorganization of the speckled nuclear domains during mitosis. Identification and control of this kinase now allows regulation of the localization of splicing factors, as well as regulation of splice site selection.